This invention is in the field of compositions which repel and prevent the stinging of marine organisms, such as jellyfish. In particular, compositions and methods for inhibiting the discharge of the stinging structure (nematocyst or polar capsule) are provided.
Swimmers and divers throughout the world are familiar with the stinging organisms of the oceans such as jellyfish, sea anemone and coral. Jellyfish stings, although seldom fatal, are a major public health problem. Lotan et al. (1992) Marine Biology 112:237-242; Lotan et al. (1994) Marine Ecology Progress Series 109:59-65. In the summer months, it is estimated that over 500,000 swimmers and divers in the Chesapeake Bay area and 200,00 persons in Florida are stung by jellyfish mainly by xe2x80x9csea nettles,xe2x80x9d the common name of Chrysaora quinquecirrha. Burnett et al. (1992) MMJ 41(6):509-513. Similarly, between the months of March and August, one in four Florida bathers are stung and more than 10,000 persons require emergency medical treatment for pruritic eruptions caused by contact with jellyfish larvae known as xe2x80x9csea lice.xe2x80x9d Tomchik et al. (1993) JAMA 269(13):1669-1672.
The members of the phylum Cnidaria (e.g., jellyfish, sea anemone and coral) and the phylum Myxozoa are all equipped with stinging subcellular organelles, known as nematocysts, cnidocysts, or polar capsules. The nematocysts are located in specialized cells (nematocytes) and consist of a capsule containing a condensed tubule with potent toxins and threads. When nematocysts discharge, the tubule penetrates into its target organism and releases its toxins. The threads arrayed on the tubule enhance the anchoring and attachment of the nematocyst tubule to its target. Thus, nematocysts are involved in target recognition, toxin delivery, infection and attachment
All members of the phyla Cnidaria and Myxozoa contain nematocysts of varying sizes, shapes and types Mariscal, pp. 129-178, xe2x80x9cNematocystsxe2x80x9d in COELENTERATE BIOLOGY: REVIEWS AND NEW PERSPECTIVES, eds. Muscatine and Lenhoff (Academic Press, New York, 1974). These different types of cnidocysts function in diverse biological roles including capture of prey, toxin delivery, recognition, attachment, adherence and infection. (see, e.g., Tardent (1995); Bioessays 17(4):351-362; Lotan et al. (1995) Nature 375:4A56; Lotan et al. (1996) Expt""l Zool. 275:444-45 1; Lotan et al., pp. 132-144, xe2x80x9cToxicology and ecology and the Mediterranean jellyfish Rhopileim nomadicaxe2x80x9d in BIOCHEMICAL ASPECTS OF MARINE PHARMACOLOGY, eds. Lazarovici et al. (Alaken, Inc., Fort Collins, Colo., 199Q); Spaulding (1972) Biol. Bull. 143:440-453; Holstein and Tardent (1984) Science 223:830-833 and Mariscal, supra. Thus, although best known for their stinging capabilities, nematocysts also play a key role in recognition, attachment and infection. For example, parasites from the phylum Myxozoa use nematocysts (polar capsules) to recognize and infect their hosts. El-Matbouli et al. (1995) J. Fish Biol. 46:919-935 and Yokoyama et al. (1995) Diseases Aquatic Organ. 21:7-11.
The main body of the nematocyte cell consists of a dense capsule, the nematocyst, within which is a high folded eversible tubule. Discharge (eversion) of this tubule is driven by the build up of a high internal hydrostatic pressure of approximately 150 atmospheres within the capsule. The eversion of the internally folded tubule occurs within 3 microseconds at accelerations of up to 40,000xc3x97g, one of the most rapid mechanical events in cell biology.
The nematocyst can be sub-divided into 3 morphological compartments with different functional entities: the capsule lumen and wall, the tubule and the sensory organelles. The capsule wall and lumen are the main components involved in developing the driving force for nematocyst discharge. The tension on the inner capsule wall during nematocyst discharge reaches up to 375 MPa. Holstein (1994) Science 265:402-404. The strong capsule wall is highly permeable to water, with a pore size of 600 Dalton. Within the resting nematocyst capsule, concentrations of up to 0.5 M of cations such as Ca++, Mg++ or K+ can be found. (see, e.g., Tardent, supra; Lubbock et al. (1981) PNAS 78(6):3624-3628; Godknecht et al. (1988) Marine Biology 100:83-92; Lubbock and Amos (1981) Nature 290(5806):500-501; Weber (1989) Int. J. Biochem. 184:465-476; Hidaka (1993) Biol. Bull. 184:97-104 and Gerke (1991) Hydrobiologia 216/217:661-669 for discussions of cations and nematocysts). The anionic counterparts are represented by poly-xcex3-L-gamma.-L-glutamatic acid (PGA) in varying degrees of polymerization. During nematocyst discharge, an extreme increase in internal capsule osmotic pressure occurs due to the influx of water. It has been suggested that the influx of water into the capsule is mediated by an internal release of the cations, such as Ca++ in sea anemone or K+ in hydra normally combined with PGA. This osmotic pressure is translated into hydrostatic pressure causing the eruption and then evagination of the tubule from the nematocyst capsule (discharge). After the nematocyst discharge, the internal cation concentration of the capsule is dissolved into the surrounding fluids.
The second compartment of the nematocyst is a highly condensed eversible tubule. This tubule serves the main role in nematocyte biological function; namely, the interaction or delivery of substances from the cnidarian or myxozoan into its target. The tubule, which is 200-850 xcexcm when elongated, is twisted more than a hundred times around its axis and is packed into the 3-10 xcexcm diameter of the nematocyst Godknecht and Tardent (1988) Marine Biol. 100:83-92. Hollow barbs, arrayed on the inner surface of the tubule, become everted during discharge and play an important role in the penetration and anchoring of the tubule into its prey. Toxins, contained on the outer surface before discharge, are delivered through the barbs after the nematocyst is anchored. Lotan et al. (1995), supra.
The sensory systems of the nematocyte are responsible for the control of nematocyst discharge. Discharge of nematocysts requires both chemical and mechanical stimulation of the sensory organelles. In the sea anemone Aiptasia pallida, two classes of chemical receptors have been identified. One type of receptor is triggered by N-acetylated sugars, while the second chemoreceptor is triggered by certain amino acids. Thorington et al. (1988) Biol Bull. 174:163-171. The mechanical sensory system is dependent on activation of the chemoreceptors. These mechanoreceptors, however, can be adjusted by the organism, for instance, by tuning them to unique frequency signals emitted by a favored prey.
Certain fish diseases, including whirling disease, are caused by infection with an obligate parasite of the phylum myxozoan. (see e.g., for descriptions of myxozoan, Yokoyama et al. (1993) Dis. Aquat. Org. 17:223-228; Yokoyama et al. (1995) Dis. Aquat. Org. 21:7-11; Smothers et al. (1994) Science 265:1719-172; and El-Matbouli et al. (1995) J. Fish Biology 46:919-935). These parasites require two hosts, a fish and an aquatic annelid. The stages emerging from each host are infectious only for the other host, however, at each infectious stage, activated polar capsules (nematocyst) release a tubule that penetrates or adheres to the integument of the target. Host recognition is species-specific, indicating that nematocysts serve an important role in recognition of their host (El-Matbouli, supra).
In sum, nematocysts provide an effective method of delivering a substance deep into the target. Because nematocysts are able to penetrate their target so efficiently, it is difficult to remove them, or to treat after the toxin has penetrated. Conventionally, nematocyst stings have been treated with antidotes such as steroids, aluminum sulfate/surfactant and antihistamines. Tomchik, supra. For example, Wasuwat (1970) Nature 225:758 describes how Thai fisherman use an extract made from the leaves of Ipomoea pea-caprae as an antidote to jellyfish poison When the extract was analyzed, it was found to be mildly antihistaminic. The extract exhibits the same effect against jellyfish poison as two commercially prepared antidotes containing antihistamines. In Australia, vinegar is recommended for treatment of lethal box jellyfish stings. Hartwick et al. (1980) Med. J. Aust. 1:15-20. Vinegar is not recommended for stings by other species. Fenner et al. (1993) Med. J. Aust. 158:498-501.
Since post-sting treatments for nematocyst stings are often unsatisfactory, the search for ways to prevent nematocyst discharge has been ongoing. The most often prescribed method of preventing jellyfish stings is avoiding any contact with the nematocysts. Tomchik, supra. However, in the case of microscopic larvae, this often means foregoing all ocean activities during the months of high incidence, (e.g., March through August in Florida). It would, therefore, be useful to have a means for inhibiting nematocyst discharge even when contact does occur.
Australian patent application 67563/94 (WO 94/17779) discloses topical hydrodispersion preparations that are reported to be effective in preventing nematocyst discharge as measured by scanning electron microscopy (SEM). The formulations contain inorganic micropigments incorporated into the lipid phase of the hydrodispersion; an optional UV filtering substance and are essentially free of emulsifiers.
Lubbock (1979) J. Exp. Biol. 83:283-292 describes how proteinaceous compounds tend to induce a stronger response leading to nematocyst discharge in sea anemones than either polysaccharides or lipids. The authors could determine no simple recognition basis and speculated that the process of nematocyst discharge was complex. Lubbock and Amos, supra, disclose that isolated nematocyst capsules do not discharge in 50 mM CaCl2. The authors report that inhibition of nematocyst discharge occurs only if a solute that could not rapidly penetrate the capsule wall is used, for example, high molecular weight polyethylene glycol. Thus, calcium in the surrounding environment may stabilize nematocysts because it reduces the differential between the calcium concentration outside the capsule and inside the capsule. Normally, the calcium concentration inside the nematocyst capsule is approximately 600 mM. Normal calcium concentration in sea water is around 7 mM, about 100 fold less than inside the capsule. Thus, increasing the calcium concentration outside the capsule to 50 mM reduces the differential to around 10 fold and may be involved a inhibiting nematocyst discharge.
Heeger et al. (1992) Marine Biology 113:669-678 tested the ability of three commercially available sunscreen lotions to inhibit jellyfish nematocyst discharge on samples of live human skin. Two of the three lotions were effective at reducing the number of nematocysts discharged. The authors concluded that glycerol and oil components of the lotions could be masking or suppressing the effects of natural stimuli of the skin, however, even the lotion which did not inhibit nematocyst discharge contained these substances. Hartwick et al. (1980) Med. J. Australia 1:15-20 report that commercial sting remedies provide do not inhibit nematocyst discharge.
Thorington et al. Biol. Bull 174:163-171 (1988) describe two classes of nematocyst chemoreceptor. One class of chemoreceptors is specific for N-acetylated sugars, and is unaffected by antihistamines. The second class of chemoreceptors is specific for amino acids and is inhibited by antihistamines. The authors hypothesized that the N-acetylated sugar receptors are the initial trigger for nematocyst discharge and that the antihistamine-affected receptors are probably activated only upon leakage of amino acids from the puncture wound created in the prey by the sugar-triggered nematocyst. It is therefore, unexpected that the present invention has shown that antihistamines alone are effective at inhibiting nematocyst discharge.
The present invention provides novel compositions that effectively inhibit nematocyst discharge. The compositions described herein are extremely effective at inhibiting nematocyst discharge when applied topically prior to exposure to nematocysts. In addition, it was the surprising discovery of the inventor that placing antihistamines and/or cations into the aqueous environment surrounding nematocyst-bearing organisms effectively inhibits nematocyst discharge. Thus, the present invention provides both protection for swimmers, divers and fishermen, as well as protection from nematocyte-facilitated infection in contexts such as fish farms where the surrounding environment can be treated.
In accordance with one embodiment of the present invention, a composition for inhibiting the discharge of nematocysts comprising an effective amount of an antihistamine is provided. In accordance with another embodiment of the present invention, compositions for inhibiting the discharge of nematocysts comprising an effective amount of at least one cation are provided. In accordance with yet another embodiment of the present invention, compositions for inhibiting the discharge of nematocysts comprising effective amounts of an antihistamine and at least one cation are also provided.
The nematocysts may be discharged from organisms selected from the group consisting of Hydrozoa, Anthozoa, Myxozoa and Schyphoza, preferably from the group consisting of Aurelia sp., Pelagia sp., Chrysaora sp., Anthoplaura sp, Rhopilema sp., Physalia sp., Cyanea sp., Linuche sp., Catostylus, Carybdea sp., Chironex sp., Stomolophus sp., Rhiozostoma and Corinactis sp., more preferably from the group consisting of Aurelia aurita, Corynactic californica, Anthopleura elegantissima, Pelagia noctiluca., Chrysaora quinquecirrha and Anthoplaura sp.
In one embodiment, the antihistamine is present in a concentration of from about 0.0005% to about 2.0%, preferably from about 0.001% to about 0.2%. The antihistamine can be any antihistamine, preferably diphenhydramine, cimetidine or tripelennamine, more preferably diphenhydramine.
In yet a further embodiment, the composition comprising an antihistamine further comprises at least one cation. The cation can be Ca++, K+, Na+, Mn++, Co++, Mg++, or Fe++ and is present in a concentration of from about 50 mM to about 1M, preferably from about 50 mM to about 500 mM, more preferably from about 50 mM to about 200 mM.
In another embodiment, the composition comprises at least one cation. The cation can be Ca++, K+, Na+, Mn++, Co++, Mg++, or Fe++ and is present in a concentration of from about 50 mM to about 1M, preferably from about 50 mM to about 500 mM, more preferably from about 50 mM to about 200 mM.
In another embodiment, compositions within the present invention are incorporated into an ointment base. In a preferred embodiment, the ointment base does not comprise a glycoprotein, and is a lipid or a silicone polymer. In yet a further embodiment, the ointment base further comprises a sunscreen.
In yet a further embodiment of the present invention, methods are provided for inhibiting nematocyst discharge comprising applying inventive compositions within the present invention to the skin of a subject prior to contact with nematocysts. In another embodiment, method are provided for inhibiting nematocyst discharge in an environment surrounding the nematocyst.
As will become apparent, preferred features and characteristics of one aspect of the invention are applicable to any other aspect of the invention.